Use of β-adrenoceptor antagonists for the manufacture of a medicament for the treatment of disorders of the outer retina

ABSTRACT

The invention is directed to the use of β-adrenoceptor antagonists for the manufacture of a medicament for the treatment of disorders of the outer retina.

This application claims priority from PCT/US00/32575 filed on Nov. 29,2000, and U.S. Ser. No. 60/167,993, filed on Nov. 30, 1999.

This invention is directed to the use of β-adrenoceptor antagonists,such as, betaxolol, for treating disorders of the outer retina.

BACKGROUND OF THE INVENTION

To date, more than 100 genes have been mapped or cloned that may beassociated with retinal degeneration. The pathogenesis of retinaldegenerative diseases such as age-related macular degeneration (ARMD)and retinitis pigmentosa (RP) is multifaceted and can be triggered byenvironmental factors in those who are genetically predisposed. One suchenvironmental factor, light exposure, has been identified as acontributing factor to the progression of retinal degenerative disorderssuch as ARMD (Young, Survey of Ophthalmology, 1988, Vol. 32:252–269).Photo-oxidative stress leading to light damage to retinal cells has beenshown to be a useful model for studying retinal degenerative diseasesfor the following reasons: damage is primarily to the photoreceptors andretinal pigment epithelium (RPE) of the outer retina (Noell, et al.,Investigative Ophthalmology & Visual Science, 1966, Vol. 5:450–472;Bressler, et al., Survey of Ophthalmology, 1988, Vol. 32:375–413;Curcio, et al., Investigative Ophthalmology & Visual Science, 1996, Vol.37:1236–1249); they share a common mechanism of cell death, apoptosis(Ge-Zhi, et al., Transactions of the American Ophthalmology Society,1996, Vol. 94:411–430; Abler, et al., Research Communications inMolecular Pathology and Pharmacology, 1996, Vol. 92:177–189); light hasbeen implicated as an environmental risk factor for progression of ARMDand RP (Taylor, et al., Archives of Ophthalmology, 1992, Vol.110:99–104; Naash, et al., Investigative Ophthalmology & Visual Science,1996, Vol. 37:775–782); and therapeutic interventions which inhibitphoto-oxidative injury have also been shown to be effective in animalmodels of heredodegenerative retinal disease (LaVail, et al.,Proceedings of the National Academy of Science, 1992, Vol.89:11249–11253; Fakforovich, et al., Nature, 1990, Vol. 347:83–86).

A number of different classes of compounds have been reported tominimize retinal photic injury in various animal models, including:antioxidants, such as, ascorbate (Organisciak, et al., InvestigativeOphthalmology & Visual Science, 1985, Vol. 26:1580–1588),dimethylthiourea (Organisciak, et al., Investigative Ophthalmology &Visual Science, 1992, Vol. 33:1599–1609; Lam, et al., Archives ofOphthalmology. 1990, Vol.108:1751–1757). α-tocopherol (Kozaki, et al.,Nippon Ganka Gakkai Zasshi, 1994, Vol. 98:948–954), and β-carotene(Rapp, et al., Current Eye Research, 1996, Vol. 15:219–223); calciumantagonists, such as, flunarizine, (Li, et al., Experimental EyeResearch, 1993, Vol. 56:71–78; Edward, et al., Archives ofOphthalmology, 1992, Vol. 109:554–622); growth factors, such as,basic-fibroblast growth factor (bFGF), brain-derived nerve factor(BDNF), ciliary neurotrophic factor (CNTF), and interleukin-1-β (LaVail,et al., Proceedings of the National Academy of Science, 1992, Vol. 89:11249–11253); glucocorticoids, such as, methylprednisolone (Lam, et al.,Graefes Archives of Clinical & Experimental Ophthalmology, 1993, Vol.231:729–736), dexamethasone (Fu, J., et al., Experimental Eye Research,1992, Vol. 54:583–594); NMDA-antagonists, such as, eliprodil and MK-801(Collier, et al., Investigative Ophthalmology & Visual Science, 1999,Vol. 40, pg. S159) and iron chelators, such as, desferrioxamine (Li, etal., Current Eye Research, 1991, Vol. 2:133–144).

Ophthalmic β-adrenergic antagonists, also referred to as β-adrenoceptorantagonists or β-blockers are well documented IOP-lowering agents fortherapy of glaucoma. Currently, several ophthalmic β-blockers areapproved for use worldwide. The majority of these are nonselectiveβ-blockers; betaxolol is a cardioselective β-blocker marketed asBetoptic® or Betoptic®S (Alcon Laboratories, Inc., Fort Worth, Tex.).

As a potential treatment for glaucoma and other inner retinapathologies, Osborne, et al. (Brain Research, 1997, Vol. 751:113–123)have shown that betaxolol is neuroprotective in a ratischemia/reperfusion injury model. Ischemia/reperfusion results in areduction of the electroretinogram (ERG) b-wave amplitude, a measure ofinner retina function, not photoreceptor or RPE function. This ERGb-wave deficit was protected by treatment with betaxolol. Consistentwith the inner retinal protection was preservation of cholineacetyltransferase and calretinin imrmunoreactivity in the innerplexiform layer and cell bodies in the ganglion cell layer and innernuclear layer by treatment with betaxolol. In vitro studies by Osborne,et al. have also shown that betaxolol can prevent the kainate inducedelevation of intracellular calcium in chick retinal cells, partiallyinhibited changes in GABA immunoreactivity in the rabbit inner retinafollowing glucose-oxygen deprivation, and partially prevented theglutamate-induced release of lactate dehydrogenase in cortical cultures.β-adrenoceptor antagonists have also been shown to relax KCl-inducedcontraction of porcine ciliary artery (Hester, et al., Survey ofOphthalmology, Vol. 38:S125–S134, 1994). Moreover, certain β-blockershave been shown to produce vasorelaxation unrelated to theirβ-adrenergic blocking action (Yu, et al., Vascular Risk Factors andNeuroprotection in Glaucoma, pp. 123–134, (Drance, S. ed.) Update, 1996;Hoste, et al., Current Eye Research, Vol. 13:483–487, 1994; and Bessho,et al., Japanese Journal of Pharmacology, Vol.55:351–358, 1991.) Thereis experimental evidence that this is due to the ability of certainβ-blockers to act as calcium channel blockers and to reduce the entry ofcalcium ion into vascular smooth muscle cells where it participates inthe contraction response and reduces the diameter of the lumen of theblood vessel and decreases blood flow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the prevention of photic retinopathy by the systemicadministration of the selective β₁-blockers, betaxolol and its isomers.

FIG. 2 shows the prevention of photic retinopathy by the systemicadministration of the non-selective β-blocker, timolol.

FIG. 3 compares the protection of the retina from photic retinopathy bybetaxolol and levobetaxolol following topical ocular administration.

FIG. 4 shows preservation of retinal function in P23H mutant rhodopsintransgenic rats.

FIG. 5 shows upregulation of endogenous retinal neurotrophic factor mRNAlevels following a single administration of levobetaxolol compared toother agents.

SUMMARY OF THE INVENTION

The present invention is directed to β-adrenoceptor antagonists whichhave been discovered to be useful in treating disorders of the outerretina, particularly: ARMD; RP and other forms of heredodegenerativeretinal disease; retinal detachment and tears; macular pucker; ischemiaaffecting the outer retina; damage associated with laser therapy (grid,focal, and panretinal) including photodynamic therapy (PDT); trauma;surgical (retinal translocation, subretinal surgery, or vitrectomy) orlight induced iatrogenic retinopathy; and preservation of retinaltransplants. As used herein, the outer retina includes the RPE,photoreceptors, Muller cells (to the extent that their processes extendinto the outer retina), and the outer plexiform layer. The compounds areformulated for systemic or local ocular delivery.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Neurotrophic factors can be potent neuroprotective agents, but aspeptides, are difficult to deliver to the retina or central nervoussystem. We have demonstrated that s betaxolol upregulates CNTF and bFGFMRNA retinal expression and this can prevent light-induced apoptoticcell death to the outer retina. We have found that treatrnent withbetaxolol can completely prevent photo-oxidative induced retinopathy andsignificantly reduce loss of retinal function. The safety advantages ofthe compound make it particularly desirable for both acute and chronictherapies. Such an agent would have utility in the treatment of variousouter retinal degenerative diseases.

In our light damage paradigms, antioxidants were either ineffective(alpha-tocopherol) or marginally effective at high doses (ascorbate,vitamin E analogs). Similarly, some calcium antagonists (flunarizine,nicardipine) were moderately effective while others (nifedipine,nimodipine, verapamil) had no effect in preventing light-inducedfunctional or morphological changes. However, it has been discoveredthat β-adrenoceptor antagonists are effective in these light damageparadigms and therefore are useful for treating disorders of the outerretina.

Disorders of the outer retina encompass acute and chronicenvironmentally induced (trauma, ischemia, photo-oxidative stress)degenerative conditions of the photoreceptors and RPE cells in normal orgenetically predisposed individuals. This would include, but not belimited to, ARMD, RP and other forms of heredodegenerative retinaldisease, retinal detachment, tears, macular pucker, ischemia affectingthe outer retina, damage associated with laser therapy (grid, focal andpanretinal) including photodynamic therapy (PDT), thermal orcryotherapy, trauma, surgical (retinal translocation, subretinal surgeryor vitrectomy) or light induced iatrogenic retinopathy and preservationof retinal transplants.

The invention contemplates the use of any β-adrenoceptor antagonist,including their isomers and pharmaceutically acceptable salts, fortreating disorders of the outer retina. Preferred β-adrenoceptorantagonists also exhibit neurotrophic activity and may have calciumantagonist activity.

Representative β-adrenoceptor antagonists useful according to thepresent invention include, but are not limited to: betaxolol (R or S orracemic), timolol, carteolol, levobunolol, metipranolol, befunolol,propranolol, metoprolol, atenolol, pendolol, and pinbutolol.

The preferred β-adrenoceptor antagonist is betaxolol, and/or its R or Sisomer. The S-isomer is also referred to as levobetaxolol.

In general, for degenerative diseases, the β-blockers of this inventionare administered orally with daily dosage of these compounds rangingbetween 0.001 and 500 milligrams. The preferred total daily dose rangesbetween 1 and 100 milligrams. Non-oral administration, such as,intravitreal, topical ocular, transdermal patch, subdermal, parenteral,intraocular, subconjunctival, or retrobulbar injection, iontophoresis orslow release biodegradable polymers or liposomes may require anadjustment of the total daily dose necessary to provide atherapeutically effective amount of the compound. The β-blockers canalso be delivered in ocular irrigating solutions used during surgery,see, for example, U.S. Pat. No. 4,443,432. This patent is hereinincorporated by reference. Concentrations should range from 0.001 μM to100 μM, preferably 0.01 μM to 5 μM.

The β-blockers can be incorporated into various types of ophthalmicformulations for topical delivery to the eye. They may be combined withophthalmologically acceptable preservatives, surfactants, viscosityenhancers, gelling agents, penetration enhancers, buffers, sodiumchloride, and water to form aqueous, sterile ophthalmic suspensions orsolutions or preformed gels or gels formed in situ. Ophthalmic solutionformulations may be prepared by dissolving the compound in aphysiologically acceptable isotonic aqueous buffer. Further, theophthalmic solution may include an ophthalmologically acceptablesurfactant to assist in dissolving the compound. The ophthalmicsolutions may contain a viscosity enhancer, such as,hydroxymethylcellulose. hydroxyethylcellulose,hydroxypropylmethylcellulose. methylcellulose, polyvinyl-pyrrolidone, orthe like, to improve the retention of the formulation in theconjunctival sac. In order to prepare sterile ophthalmic ointmentformulations, the active ingredient is combined with a preservative inan appropriate vehicle, such as, mineral oil, liquid lanolin, or whitepetrolatum. Sterile ophthalmic gel formulations may be prepared bysuspending the active ingredient in a hydrophilic base prepared from thecombination of, for example, carbopol-940, or the like. according to thepublished formulations for analogous ophthalmic preparations;preservatives and tonicity agents can be incorporated.

If dosed topically, the β-blockers are preferably formulated as topicalophthalmic suspensions or solutions, with a pH of about 4 to 8. Theβ-blockers will normally be contained in these formulations in an amount0.001% to 5% by weight, but preferably in an amount of 0.01% to 2% byweight. Thus, for topical presentation, 1 to 2 drops of theseformulations would be delivered to the surface of the eye 1 to 4 timesper day according to the discretion of a skilled clinician.

The preferred β-blocker, betaxolol (or its R or S isomer), is orallybioavailable, demonstrates a low incidence of adverse effects uponadministration, and effectively crosses the blood-brain barrierindicating that effective concentrations are expected in the targettissue, the retina. Betaxolol is described in U.S. Pat. Nos. 4,252,984and 4,311,708, the contents of which are incorporated herein byreference.

β-adrenoceptor antagonists were evaluated in our photo-oxidative inducedretinopathy paradigm, a model of retinal degenerative diseases that mayhave utility for identifying agents for treatment of RP and ARMD.Unexpectedly betaxolol and its enantiomers, demonstrated marked potencyand efficacy as a neuroprotective agent. Both photoreceptor and RPEcells were completely protected from light-induced functional changesand morphologic lesions. Timolol was also neuroprotective, but wassignifiantly less potent. Additional evaluation of levobetaxolol in atransgenic rat model that has a rhodopsin mutation, which is similar toa defect observed in some human patients with heredodegenerativedisease, provided significant protection of retinal function.

EXAMPLE 1 Prevention of Photo-oxidative Induced Retinopathy by Betaxololand its Enantiomers

Photic retinopathy results from excessive excitation of the RPE andneuroretina by absorption of visible or near ultraviolet radiation.Lesion severity is dependent upon wavelength, irradiance, exposureduration, species, ocular pigmentation, and age. Damage may result fromperoxidation of cellular membranes, inactivation of mitochondrialenzymes such as cytochrome oxidase, and/or increased intracellularcalcium. Cellular damage resulting from photo-oxidative stress leads tocell death by apoptosis (Shahinfar, et al., 1991, Current Eye Research,Vol. 10:47–59; Abler, et al., 1994, Investigative Ophthalmology & VisualScience, Vol. 35(Suppl):1517). Oxidative stress induced apoptosis hasbeen implicated as a cause of many ocular pathologies, including,iatrogenic retinopathy, macular degeneration, RP and other forms ofheredodegenerative disease, ischemic retinopathy, retinal tears retinaldetachment, glaucoma and retinal neovascularization (Chang, et al.,1995, Archives of Ophthalmology, Vol. 113:880–886; Portera-Cailliau, etal., 1994, Proceedings of National Academy of Science (U.S.A.), Vol.91:974–978; Buchi, E. R., 1992, Experimental Eye Research, Vol.55:605–613; Quigley, et al., 1995, Investigative Ophthalmology & VisualScience, Vol. 36:774–786). Photic induced retinal damage has beenobserved in mice (Zigman, et al., 1975, Investigative Ophthalmology &Visual Science, Vol. 14:710–713), rats (Noell, et al., 1966,Investigative Ophthalmology and Visual Science, Vol. 5:450–473;Kuwabara, et al., 1968, Archives of Ophthalmology, Vol. 79:69–78;LaVail, M. M., 1976, Investigative Ophthalmology & Visual Science, Vol.15:64–70), rabbit (Lawwill, T., 1973, Investigative Ophthalmology &Visual Science, Vol. 12:45–51), and squirrel (Collier, et al., 1989; InLaVail et al., Inherited and Environmentally Induced RetinalDegenerations, Alan R. Liss, Inc., New York; Collier, et al., 1989,Investigative Ophthalmology & Visual Science, Vol. 30:631–637),non-human primates (Tso, M. O. M., 1973, Investigative Ophthalmology &Visual Science, Vol. 12:17–34; Ham, et al., 1980, Vision Research, Vol.20:1105–1111; Sperling, et al., 1980, Vision Research, Vol.20:1117–1125; Sykes, et al., 1981, Investigative Ophthalmology & VisualScience, Vol. 20:425–434; Lawwill, T., 1982, Transactions of theAmerican Ophthalmology Society, Vol. 80:517–577), and man (Marshall, etal., 1975, British Journal of Ophthalmology, Vol. 59:610–630; Green, etal., 1991, American Journal of Ophthalmology, Vol. 112:520–27). In man,chronic exposure to environmental radiation has also been implicated asa risk factor for ARMD (Young, R. W., 1988, Survey of Ophthalmology,Vol. 32:252–269; Taylor, et al., 1992, Archives of Ophthalmology, Vol.110:99–104; Cruickshank, et al., 1993, Archives of Ophthalmology, Vol.111:514–518).

Systemic Dosing

The purpose of Experiment 1 was to determine if selective β-adrenoceptorantagonists, in particular betaxolol (racemic), levobetaxolol(S-isomer), and betaxolol (R-isomer) are neuroprotective and can rescueretinal cells from a photo-oxidative induced retinopathy. The purpose ofExperiment 2 was to determine the dose-dependent efficacy of timolol, apotent non-selective β₁- and β₂-blocker, in this photo-oxidative stressmodel. Male Sprague Dawley rats were randomly assigned to drug orvehicle experimental groups. Rats received three intraperitoneal (IP)injections of either vehicle or drug at 48, 24, and 0 hours prior to a6-hour light exposure to spectrally filtered blue light (˜220 fc).Control rats were housed in their home cage under normal cyclic lightexposure. Control rats were not dosed with either vehicle or drug. TheERG is a non-invasive clinical measurement of the electrical response ofthe eye to a flash of light. The a-wave and b-wave are two components ofthe ERG that are diagnostic of retinal function. The a-wave reflectsouter retina function and is generated by interactions betweenphotoreceptor and RPE while the b-wave reflects inner retina function,particularly on-bipolar cells. Although the inner retina is notsignificantly damaged by this light exposure, the b-wave is depresseddue to the lack of photoreceptor input. Changes in the a-wave amplitudeor latency are diagnostic of outer retina pathology. The ERG wasrecorded after a five day recovery period from dark-adapted anesthetizedrats (ketamine-HCl, 75 mg/Kg; xylazine, 6 mg/Kg). The eye's electricalresponse to a flash of light was elicited by viewing a ganzfeld. ERGs toa series of light flashes increasing in intensity were digitized toanalyze temporal characteristics of the waveform and responsevoltage-log intensity relationship.

Results Experiment 1: Comparison of Betaxolol with its R and S Isomer

Vehicle Dosed Rats. Blue-light exposure for 6 hours resulted in asignificant diminution of the ERG response amplitude (ANOVA, p<0.001)compared to controls when measured after a 5-day recovery period (FIG.1). Maximum a-wave and b-wave amplitudes were reduced approximately 66%in vehicle-dosed rats compared to controls. In addition, thresholdresponses were lower and evoked at brighter flash intensities.Betaxolol (racemic). Systemic (IP) dosing with betaxolol (racemic)provided dose-dependent protection of outer and inner retina functionagainst this light-induced retinal degeneration in rats after a 5-dayrecovery period (FIG. 1). Maximum a-wave response amplitudes inbetaxolol dosed rats with 20 and 40 mg/kg were 1.9 and 2.1 fold higher,respectively, than vehicle dosed rats.Levobetaxolol (S-isomer). Systemic administration of levobetaxololprovided dose-dependent protection of outer retina function when theERGs were measured 5 days after induction of this severe photo-oxidativeinduced retinopathy. Systemic dosing with 20 mg/kg and 40 mg/kglevobetaxolol afforded significant protection of retinal function tothis oxidative insult (FIG. 1). ERG amplitudes in rats dosed with 20mg/kg were 69% of normal and twice the amplitude of vehicle-dosed rats.Complete protection of the retinal response to a flash of light wasmeasured after a 5-day recovery period in rats dosed with levobetaxolol(40 mg/kg). This protection persisted after a 4-week recovery period.Betaxolol (R-isomer). Partial but significant protection of outer andinner retina function against light-induced retinal degeneration wasmeasured in rats dosed with 20 and 40 mg/kg (FIG. 1). ERGs wereapproximately 64% of normal in rats dosed (20 or 40 mg/kg) with theR-isomer of betaxolol. This protection persisted after a 4-week recoveryperiod.

Experiment 2: Prevention of Photic Retinopathy by Timolol

Five days after blue-light exposure, outer retinal function in vehicledosed rats was reduced by 54% and inner retina function was reduced 52%(FIG. 2). Systemic administration (IP) of tirnolol at 10, 20, and 40mg/kg afforded no significant protection of retinal function to thisphoto-oxidative insult (FIG. 2). ERGs recorded from rats dosed with 80mg/kg were significantly better than responses measured in vehicle dosedrats.

Conclusion

Systemic administration of the β-adrenoceptor antagonists, betaxolol andits enantiomers, provided dose-dependent neuroprotection of outer andinner retina function when measured 5-days or 4-weeks after induction ofa severe photo-oxidative induced retinopathy. Significant retinalprotection was measured in rats dosed with these β-adrenoceptorantagonists at 20 and 40 mg/kg. This photic-induced retinopathy wasprevented in rats dosed with levobetaxolol. Timolol, a non-selectiveβ-blocker, was also effective in reducing the severity of oxidativedamage to the retina as a result of this light exposure.

EXAMPLE 2 Prevention of Photo-oxidative Induced Retinopathy by TopicalOcular Dosing with Levobetaxolol

The purpose of this experiment was to determine the degree of retinalprotection that could be measured in rats following topical oculardosing. Levobetaxolol (0.5%), (racemic) betaxolol (0.5%), and vehiclewere evaluated in the photic retinopathy model. Induction ofphotochernical lesions and evaluation of retinal function with the ERGwere performed as described in the photo-oxidative induced retinopathyparadigm used in Example 1.

Subjects and Dosing

Male Sprague Dawley rats were randomly assigned to either a vehicledosed group (N=10), (racemic) betaxolol (0.5%) dosed group (N=10) orlevobetaxolol (0.5%) dosed group (N=10). Rats were dosed topical ocular(b.i.d.) with two drops per eye. Rats were pre-dosed for 17 days priorto light exposure and dosed an additional two days after the lightexposure. Control rats (N=4) were housed in their home cage under normalcyclic light exposure.

Results

Blue-light exposure to vehicle dosed rats resulted in a significantreduction in retinal function (ANOVA, p<0.004), as measured by theelectroretinogram (ERG), when measured five days after light exposure(FIG. 3). Maximum a-wave response amplitudes were reduced by 58% andinner retina function was reduced 56%.

Topical ocular dosing with levobetaxolol (b.i.d.) provided significantprotection when compared to vehicle dosed rats (FIG. 3). Further,levobetaxolol completely ameliorated this photic induced retinopathy asno significant difference in retinal function was detected betweencontrol and levobetaxolol dosed rats.

No significant protection was measured in betaxolol (racemic) dosedrats. In betaxolol dosed rats, ERG response amplitudes were higher butnot significantly different than responses measured from vehicle dosedrats.

EXAMPLE 3 Preservation of Visual Function in Transgenic Rats byLevobetaxolol

The P23H rhodopsin mutated transgenic rat has a specific rhodopsinmutation that has been identified in subsets of patients with RP. Thisdegeneration is characterized by a slow degeneration of retinalphotoreceptors and marked reduction in the electroretinogram. As inlight damage, photoreceptor loss is primarily through an apoptoticprocess.

Methods Subjects and Dosing

At the time of weaning, rats are randomly assigned to either a drug orvehicle group. Rats were dosed (oral gavage) with vehicle orlevobetaxolol (40 mg/kg,) every other day. This dose was evaluated basedon its ability to completely ameliorate a photic induced retinopathy.ERGs were recorded as described in Example 1.

Results

Oral dosing with levobetaxolol (40 mg/kg) every other day significantlyattenuated the loss of retinal function measured in 3- and 6-month oldP23H mutant rhodopsin transgenic rats compared to vehicle dosed rats(FIG. 4). Outer retinal function in 6-month old rats was 32% better thanresponses measured in vehicle dosed rats.

EXAMPLE 4 Upregulation of Retinal Endogenous Neurotrophic Factors byBetaxolol.

LaVail and others (Faktorovich, et al, Nature, Vol. 347:83–86, 1990;LeVail, et al., Proceedings of the Naional Academy of Science, 1992,Vol. 89:11249–11253), have shown that intravitreal injection of a numberof growth factors can prevent light damage to the retina. Theseneurotrophic factors are large peptides and don't easily cross theblood-retinal barrier. In terms of a therapeutic strategy for treatmentof chronic degenerative retinal disease, repeated intravitrealinjections potentially present complications, including hemorrhage,retinal detachment, and inflammation. An alternative strategy is the useof adenovirus-mediated gene transfer (bFGF in the RCS rat, Cayouette, etal, Journal of Neuroscience, Vol. 18(22):9282–93, 1998, and CNTF in therd mouse, Cayouette, et al., Human Gene Therapy, Vol. 8(4):423–30,1997), which has had limited success in preventing photoreceptor lossdue to loss of expression over time and non-homogeneous infection ofcells. We have shown that placement of genetically engineered cells intothe vitreous that secrete CNTF are also effective in preventing anoxidative induced retinopathy. A recent strategy has been to identifypharmacologic agents that upregulate endogenous growth factors. Wen etal, (WO 98/10758, Mar. 19, 1998), have shown that α₂-adrenoceptoragonists can upregulate bFGF and prevent photic injury. To determine ifa β-adrenergic antagonist can induce endogenous production ofneurotrophic factors, levobetaxolol was evaluated.

Evaluation of Levobetaxolol

Male albino Sprague Dawley rats were given a single IP injection ofeither an α₂-adrenoceptor agonist (brimonidine) (20 mg/kg), aβ-adrenergic antagonist (levobetaxolol) (20 mg/kg), or vehicle andmaintained in the dark for 12 hours prior to harvesting of retinaltissue. Dark-adapted normal control rats were also evaluated. Endogenousretinal growth factor mRNA upregulation was determined by Northern blotanalysis. Retinas were flash frozen in liquid nitrogen and stored untilisolation of total RNA. RNA samples were run on a 1.2% agarose gel,transferred to nylon membranes, prehybridized, hybridized with labeledcDNA probes for 16 hours, washed, and exposed to X-ray film. The blotswere then stripped and reprobed with an oligo specific for the 18S RNA.The bands specific for bFGF, CNTF and 18S RNA were scanned in a gelimage scanner and analyzed.

RESULTS

No difference was observed in the bFGF/18S or CNTF/18S ratio betweenvehicle dosed and control rats (FIGS. 5).

A single dose of brimonidine (20 mg/kg) resulted in a 14 fold increasein bFGF mRNA expression (FIG. 5). However, CNTF mRNA expression was notupregulated in these rats.

Similarly, levobetaxolol, a β-adrenergic antagonist, induced a 13-foldincrease in bFGF mRNA expression in rats receiving a single IP injection(20 mg/kg) (FIG. 5). In addition to upregulating bFGF in these rodentretinas. endogenous CNTF mRNA expression was upregulated by a factor of2.3 compared to background expression. Treatment with recombinant-CNTFhas been shown to be efficacious in prevention of photic retinopathy andretinal heredodegenerative change.

CONCLUSION

We unexpectedly found that levobetaxolol was a potent inducer ofendogenous bFGF mRNA. Unlike α-adrenoceptor agonists, levobetaxolol alsoresulted in a marked elevation of CNTF mRNA expression. Further, we havedemonstrated that dosing with levobetaxolol, betaxolol (racemic) or itsR-isomer provided significant protection to the retina when stressedwith a severe photo-oxidative insult. The upregulation of CNTF mRNA isparticularly important in treatment of retinopathy. The efficacy of CNTFor its analogue in preventing outer retinal degeneration has beendemonstrated in the rat and mouse phototoxicity model, RCS dystrophicrat, Rdy cat suffering a rod-cone dystrophy, retinal degeneration caninemodel, transgenic rat (P23H and Q344ter), transgenic mouse (Q344ter), rdmouse and rds mouse. On the other hand, bFGF has only demonstratedefficacy in the rat and mouse phototoxicity model and RCS dystrophicrat.

Based on these novel findings we conclude that β-adrenoceptorantagonists, in particular levobetaxolol and betaxolol, areneuroprotective in transgenic rat and photo-oxidative stress models(FIGS. 1, 2, 3, and 4) and would be effective in the treatment ofvarious ophthalmic degenerative diseases of the outer retina.Neuroprotection may be afforded by upregulation of endogenousneurotrophic factors. including, CNTF and bFGF (FIG. 5).

EXAMPLE 5

Levobetaxolol Hydrochloride Formulations Concentration 0.25% 0.5% 0.75%Ingredient Percent w/v Percent w/v Percent w/v Levobetaxololhydrochloride 0.28^(a) 0.56^(b) 0.84^(c) Poly(styrene 0.375 0.75 1.125divinylbenzene) Sulfonic Acid Carbomer 974 P 0.35 0.35 0.35 Mannitol 4.54.0 3.67 Boric Acid 0.3 0.3 0.3 Disodium Edetate 0.01 0.01 0.01Benzalkonium Chloride 0.01 + 0.01 + 0.01 + 5% excess^(d) 5% excess^(d)5% excess^(d) N-Lauroylsarcosine 0.03 0.03 0.03 Tromethamine pH adjustpH adjust pH adjust to 6.5 to 6.5 to 6.5 Hydrochloric Acid 6.5 ± 0.2 6.5± 0.2 6.5 ± 0.2 (if needed) Purified Water qs 100% qs 100% qs 100%^(a)Equivalent to 0.25% betaxolol free base ^(b)Equivalent to 0.5%betaxolol free base ^(c)Equivalent to 0.75% betaxolol free base ^(d)The5% excess is added as an overage

EXAMPLE 6

Betoptic ® S Betaxolol Ophthalmic Ophthalmic Ingredient Suspension,0.25% Suspension Racemic Betaxolol 0.28 + 5% xs 0.28 Poly(styrenedivinylbenzene 0.25 0.25 Sulfonic Acid) Carbomer 974P 0.2 0.45 Mannitol4.5 4.5 Boric Acid — 0.4 Edetate Disodium 0.01 0.01 BenzalkoniumChloride 0.01 + 10% excess 0.01 + 5% excess N-Lauroylsarcosine — 0.03Tromethamine and, if needed, Adjust pH 7.6 ± 0.2 Adjust pH 7.0 ± 0.2Hydrochloric Acid Purified Water qs 100 qs 100

1. A method for treating disorders of the outer retina in a patient inneed thereof, which comprises administering a pharmaceutically effectiveamount of a β-adrenoceptor antagonist selected from the group consistingof: betaxolol (R or S or racemic), timolol, carteolol, levobunolol,metipranolol, befunolol, propranolol, metoporolol, atenolol, pendolol,and pinbutolol, wherein said disorder is selected from the groupconsisting of ARMD; RP; retinal detachment and tears; macular pucker;ischemia affecting the outer retina; damage associated with lasertherapy (grid, focal and panretinal) including photodynamic therapy,thermal or cryotherapy; damage resulting from trauma to the eye;surgical (retinal translocation, subretinal surgery or vitrectomy) orlight-induced iatrogenic retinopathy; and preservation of retinaltransplant.
 2. The method of claim 1 wherein the β-adrenoceptorantagonist is betaxolol or its R or S isomer.